Cholesterol and other sterols are natural constituents of many foodstuffs. However, the presence of large amounts of cholesterol and other sterols in the human body is considered by physicians to be deleterious, since cholesterol has been implicated as a factor in a number of diseases, especially atherosclerosis, in which deposits containing a high proportion of cholesterol are deposited in blood vessels. Accordingly, it is common practice for physicians to recommend to patients who have suffered a heart attack, or who display a likelihood of, or documented, hypercholesterolemia, that the patients reduce their cholesterol intake from foodstuffs.
However, cholesterol is found in significant quantities in a wide variety of foodstuffs, being present in most animal fats, and consequently restrictions upon the cholesterol intake of patients necessitate prohibiting or greatly reducing the consumption of many foodstuffs, a step which many patients are reluctant to take, and which may introduce complications in ensuring that the patients receive a properly balanced diet meeting all nutritional requirements. Moreover, cholesterol is present in large amounts in egg yolks. Eggs are used in some processed food applications and formulas as a binding agent, and if the eggs are eliminated it is difficult to produce a foodstuff having the expected texture. Finally, the public has recently become increasingly aware of the health risks associated with consumption of cholesterol, so that even persons who are not under medical treatment for conditions in which cholesterol is implicated are voluntarily attempting to reduce their cholesterol consumption, and the food industry is beginning to label foods to show their cholesterol content. Thus, many people may avoid foods known to be high in cholesterol and seek substitutes.
In order to help people to reduce their cholesterol consumption without major modifications in their diet (and thus help to ensure, inter alia, that people who must follow a low cholesterol diet for medical reasons do in fact keep to such a diet), it is desirable to provide some method by which cholesterol and other sterol compounds (many of which can be metabolized to cholesterol or its derivatives) can be extracted from various foodstuffs, thereby producing low-cholesterol versions of such foodstuffs which can be consumed in place of the original, high-cholesterol foodstuffs. However, the requirements for such a sterol-removal process are exacting. The process must not, of course, introduce into the foodstuff any material which is not generally recognized as safe for use in foodstuffs. The process should remove from the foodstuff not only cholesterol itself but also cholesterol derivatives and other sterol compounds which can be metabolized in the body to cholesterol or derivatives thereof, and which thus affect cholesterol levels in the body. Furthermore, the process should leave the foodstuff in a form which is as close as possible to that of the original, high-cholesterol foodstuff. For example, when used on egg yolks, the cholesterol-removal process should maintain the high viscosity of the egg yolks since this high viscosity is needed for proper binding action when the treated egg yolk is used in the production of baked goods, liquid egg substitutes, margarines etc., and should not denature the proteins present in the egg yolk. Finally, the cholesterol-removal process should preserve the nutritional value of the foodstuff, and not, for example, remove vitamins and other important constituents of the foodstuff. In particular, since cholesterol is frequently present in foodstuffs in the form of various complexes, it is desirable that a cholesterol-removal process not remove the other natural materials found to be associated with the cholesterol.
Numerous attempts have previously been made to provide a cholesterol-removal process which meets these exacting criteria. For example, attempts have been made to remove cholesterol, and other undesirable food components, by extracting the cholesterol from the foodstuff with liquid, usually supercritical, carbon dioxide. Such carbon dioxide extraction processes suffer from the disadvantage that they must be operated under pressure to keep the carbon dioxide in the liquid phase, which increases the cost of the apparatus required. In addition, such carbon dioxide extraction processes may not be very selective in removing cholesterol, and thus may remove valuable constituents of the foodstuff. In addition, the properties of some foodstuffs may be altered disadvantageously by contact with liquid carbon dioxide; for example, in some cases the carbon dioxide may remove flavoring and/or odiferous components, thereby affecting the taste and/or smell of the treated foodstuff.
For example, U.S. Pat. No. 4,692,280, issued Sept. 8, 1987, to Spinelli et al., describes a process for the purification of fish oils in which the oil is extracted with supercritical carbon dioxide to remove cholesterol, together with odoriferous and volatile impurities.
Food Science and Technology Abstracts, 6, Abstract 8 A 374 (1974) (Abstract of Food Technology 28(6), 32-34, 36, 38 (1974)) describes a pilot plant for extraction of volatile substances from liquid and solid foods using liquid carbon dioxide as the solvent.
Food Science and Technology Abstracts, 18(1), Abstract 1 H 46 (1986) (Abstract of German Offenlegungsschrift 33 31 906 (1985)) describes extraction of caffeine from coffee beans using supercritical carbon dioxide as the solvent.
Food Science and Technology Abstracts, 18(2), Abstract 2 M 118 (1986) (Abstract of Agricultural and Biological Chemistry, 49(8), 2367-72 (1985)) describes extraction of oils from wheat germ using supercritical carbon dioxide as the solvent.
Food Science and Technology Abstracts, 18(3), Abstract 3 T 70 (1986) (Abstract of Indian Food Industry, 3(2), 48-51 (1084)) describes extraction of flavor components from natural products using both liquid and supercritical carbon dioxide.
Food Science and Technology Abstracts, 18(8), Abstract 8 V 321 (1986) (Abstract of French Patent Application Publication No. 2,563,702 (1985)) describes extraction of essential oils from blackcurrant buds using supercritical carbon dioxide.
Food Science and Technology Abstracts, 19(3), Abstract 3 V 102 (1987) (Abstract of United Kingdom Patent Application Publication No. 2,173,985 (1986)) describes extraction of aroma materials from dried plant material, which has been milled and soaked in ethanol, using a continuously flowing stream of carbon dioxide at a temperature below its critical temperature. The plant material can be used for extraction of tannin, caffeine and nicotine from tea, coffee and tobacco respectively.
Food Science and Technology Abstracts, 19(4), Abstract 4 E 11 (1987) (Abstract of Food Manufacture, 61(12), 58 (1986)) describes the use of liquid, supercritical or high pressure carbon dioxide in various processes, including decaffeination of coffee, preparation of hop extract for brewing, extraction of essential oils, defatting of potato chips, and fractionation of fish oils.
Food Science and Technology Abstracts, 19(4), Abstract 4 N 36 (1987) (Abstract of Seafood Export Journal 18(9), 10-13 (1986)) describes extraction of oils from Antarctic krill using supercritical carbon dioxide.
Food Science and Technology Abstracts, 19(6), Abstract 6 G 29 (1987) (Abstract of Nahrung 30(7), 667-671 (1986)) describes defatting of baker's yeast protein extracts by extraction with supercritical carbon dioxide.
Food Science and Technology Abstracts, 19(12), Abstract 12 H 200 (1987) (Abstract of Journal of Food Science and Technology 23(6), 326-328 (1986)) describes decaffeination of coffee using supercritical carbon dioxide as solvent.
Food Science and Technology Abstracts, 20(2), Abstract 2 E 35 (1988) (Abstract of Voedingsmiddelentechnologie 20(7), 32-35 (1987)) describes various uses of extraction with supercritical carbon dioxide in the food industry, including extraction of oils and fats, preparation of hop extracts, fractionation of oils and fats, extraction of essential oils, and elimination of undesirable constituents, for example decaffeination of coffee.
Food Science and Technology Abstracts, 20(3), Abstract 3 N 31 (1988) (Abstract of Agricultural and Biological Chemistry, 51(7), 1773-77 (1987)) describes fractional extraction of rice bran oil with supercritical carbon dioxide.
Food Science and Technology Abstracts, 20(4), Abstract 4 E 36 (1988) (Abstract of Food Trade Review 57(9), 461, 463-464 (1987)) describes the use of supercritical carbon dioxide as an extractant of vegetable oils.
Swientek, Supercritical fluid extraction separates components in foods, Food Processing 48(7), 32, 34, 36 (1987)) describes the use of supercritical fluid extraction in the food industry, including removal of cholesterol from milkfat, extraction of omega-3-fatty acids from fish oil, and extraction of oil seeds.
Food Science and Technology Abstracts, 20(5), Abstract 5 T 58 (1988) (Abstract of Sciences des Aliments 7(3), 481-498 (1987)) describes the preparation of a black pepper oleoresin by extraction of the pepper with supercritical carbon dioxide or with a carbon dioxide/ethanol blend.
Food Science and Technology Abstracts, 20(6), Abstract 5 T 58 (1988) (Abstract of West German Patentschrift 30 11 185 (1988)) describes the purification of lecithin for food or pharmaceutical use by extraction with supercritical carbon dioxide.
Food Science and Technology Abstracts, 20(7), Abstract 7 N 60 (1988) (Abstract of Journal of the American Oil Chemists' Society 65(1), 109-117 (1988)) describes fractionation of menhaden oil ethyl esters using supercritical fluid carbon dioxide to produce cholesterol-rich and cholesterol-depleted fractions.
Food Science and Technology Abstracts, 20(8), Abstract 8 E 4 (1988) (Abstract of Bio/Technology 6(4), 393-394, 396 (1988)) describes industrial scale use of supercritical fluid extraction with retrograde condensation to recover the condensation to recover the solute. Applications of this technology include extraction of caffeine from coffee, removal of toxic thujone from wormwood flavoring, extraction of triacylglycerols from many sources, extraction of sterols and steroids from poultry and meat products, and extraction of essential oils from thyme.
Food Science and Technology Abstracts, 20(8), Abstract 8 N 26 (1988) (Abstract of Energy in Agriculture 6(3), 265-271 (1987)) describes extraction of peanut oil using supercritical carbon dioxide.
Food Science and Technology Abstracts, 20(12), Abstract 12 N 16 (1988) (Abstract of Dissertation Abstracts International, B 48(9), 2632 (1988)) describes extraction of oil from Canola (Brassica naous or B. campestris) seed using supercritical carbon dioxide.
In addition to the problems previously mentioned, prior art processes for extraction of cholesterol and other components from foodstuffs using liquid or supercritical carbon dioxide normally involve high energy costs, since not only is the carbon dioxide itself costly, but before the carbon dioxide can be recycled to treat further batches of the foodstuff, the dissolved cholesterol is removed by allowing the carbon dioxide to evaporate (technically speaking, supercritical carbon dioxide is simply decompressed) to produce gaseous carbon dioxide and a liquid or solid residue, and the gaseous carbon dioxide must then be recompressed (and if necessary liquified) to produce liquid or supercritical carbon dioxide; this recompression is energy intensive. Accordingly, the cost of extraction of cholesterol from foodstuffs using liquid or supercritical carbon dioxide could be reduced if a way could be found to remove cholesterol from the carbon dioxide without the need to evaporate and recompress this material. This invention provides such a process for removal of cholesterol or other sterol compounds from carbon dioxide or other solvent laden with these sterols.
Furthermore, a wide variety of techniques have previously been employed in the extraction of materials from, and the purification of, complex organic mixtures, and examples of such techniques will now be given.
Food Science and Technology Abstracts, 20(11), Abstract 11 V 36 (1988) (Abstract of International Patent Application Publication No. WO 88/02989 (1988)) describes a process for the simultaneous deodorization and cholesterol reduction of fats and oils by deaeration, mixing with steam, heating, flash vaporizing, thin-film stripping with countercurrent steam, and cooling (all the preceding steps being performed under vacuum), and storage under oxygen-free conditions. This process demonstrates the difficulty in removing cholesterol from a foodstuff while maintaining the expected flavor thereof.
Deutsch et al., "Isolation of Lipids from plasma by Affinity Chromatography", Biochemical and Biophysical Research Communications, 50(3), 758-764 (1973) describes the extraction of certain lipid fractions from plasma by affinity chromatography. Cross-linked agarose (SEPHAROSE 4B) was activated by the well known cyanogen bromide method of Cuatrecasas. Dodecylamine was then covalently bound to the activated agarose to provide the adsorbent. Plasma was mixed with the adsorbent, whereupon the adsorbent was then filtered and washed. Lipids were then eluted off the adsorbent with ethanol. This procedure removed approximately 50% of the triglycerides and nearly all of the cholesterol and lipoproteins. This procedure of would be expected to work on dairy products such as milk. However, milk products require triglycerides and lipoproteins for integrity, mouthfeel and taste. For example, the fat in butter is necessary for cooking. Accordingly, this procedure would alter milk products such that much of the fat and nutrients would be removed.
U.S. Pat. No. 4,431,544 to Atkinson et al. teaches a high pressure liquid affinity chromatography by which biomolecules are extracted from solution and purified. Ligands are attached to matrices by way of spacer arms to provide the adsorbent. The matrix may be cross-linked agarose, and the spacer arms may be polyarginine or polylysine. The extraction of cholesterol from dairy products through this general-ligand affinity chromatography process is not feasible because of the broad specificity, and the toxic nature of the crosslinking agents. For example, cyanogen bromide is recommended for crosslinking a diaminoalkane spacer arm to cross-linked agarose. Cyanogen bromide is a well known cross-linking agent; however, cyanate groups are formed on the agarose hydroxyl groups not bound to spacer arms or ligands.
There is evidence that all systems using cyanogen bromide for coupling result in a significant degree of solubilization or leakage of the immobilized ligand. Parikh and Cuatrecasas discuss the problems associated with cyanogen bromide in their paper "Affinity Chromatography," Parikh et al., Chemical & Engineering News, Aug. 26, 1985, pages 17-32. Single point attachment of the ligand can result in a leakage of 1 ppm. Leakage can be reduced but evidently not eliminated. While the level of cyanide salts is less than the lethal dose of 0.1 milligrams percent, the possibility of cyanide contamination in food products should be avoided.
Heterogeneous mixtures of biomolecules may also be separated by differential migration chromatography, in which separation is effected by the differential migration of molecules through a filter material. The solute molecules migrate through the filter material at different rates due to different attractions occurring between the filter material and charges and/or functional groups on the solute molecules; the solute molecules are not actually retained on the filter material.
U.S. Pat. No. 4,544,485 to Pinkerton et al. teaches a high-pressure liquid chromatography process in which the packing material discriminates between analyte species on the basis of their different interactions with hydrophobic internal surfaces versus hydrophilic external surfaces. The hydrophobic surface may have lysine or arginine covalently bound to glyceroylpropyl groups on the support packing surface via hydroxy functionalities. The material is useful for separating small hydrophobic molecules (e.g., drugs) from protein-containing biological matrices.
U.S. Pat. No. 4,076,930 to Ellingboe teaches a column packing material which may be used to separate cholesterol, among other molecules. The material comprises hydroxyalkyl ethers of hydroxyalkoxy polysaccharides. Hydrocarbon radicals attached by ether linkages confer strongly lipophilic solvation characteristics.
U.S. Pat. No. 3,814,255 to Smernoff teaches a triglyceride cholesterol analysis in which the column material comprises activated porous inorganic oxide particles.
U.S. Pat. No. 3,817,706 to Smith teaches a fluorescence quantitative thin layer chromatographic method in which an adsorbent such as alumina, silica acid or silica gel is used on a plate to separate analytes including cholesterol. These analytes are stained and quantified.
U.S. Pat. No. 3,997,298 to McLafferty et al. teaches a ligand chromatography-mass spectrometry system and method. Quantitative and qualitative analysis of analytes, including cholesterol, is effected using a system coupling a liquid chromatography column to a mass-spectrometer chemical ionization detector.
Netherlands Patent Application No 8304501A to Utrecht teaches a column structure for a high-pressure liquid chromatography procedure. Steroids and lipids may be separated.
U.S. Pat. No. 3,527,712 to Renn et al. teaches a dried agarose gel, a method of preparation thereof and production of an aqueous agarose gel. A dissolved macro-molecular hydrocolloid is introduced into the porous structure of an agarose gel. The hydrocolloid may include cellulose derivatives, amides or polysaccharides. The material is useful for sorting molecules having molecular weights greater than 200,000, when present at concentrations of less than 5 percent. Separation of smaller molecules than molecular weight 200,000 is possible when the material is present at concentrations greater than 5 percent.
"Sephadex Column Chromatography as an Adjunct to Competitive Protein Binding Assays of Steroids," Nature New Biology, 232, 21-24 (July 1971), teaches using a column packing material comprising SEPHADEX LH-20 to separate heterogeneous mixtures of steroids. The alkylation of the hydroxyl groups of SEPHADEX makes it possible to elute with organic as well as aqueous solvents.
"Evaluation of a High-Performance Liquid Chromatography Method for Isolation and Quantitation of Cholesterol and Cholesterol Esters," Carroll et al., J. Lipid Res, 22(2), 359-363 (Feb. 1981), discusses using high pressure liquid chromatography for analyzing cholesterol.
Differential migration chromatographic techniques, including those outlined above, provide high resolution separation of solute materials. With this procedure it is possible to separate closely related compounds and thus enable qualitative and quantitative analysis of these compounds; however, such techniques are not commercially feasible for the extraction of cholesterol from foodstuffs because too many different solutes would be separated, the foodstuff would be highly diluted, and post-treatment of the foodstuff filtrate would be cumbersome.
Various potential methods for the separation of cholesterol from foodstuffs, including the affinity chromatography methods discussed above, depend upon the selection of a material which has a strong affinity for cholesterol. A number of substances are known to have such an affinity. These include macromolecular carrier proteins, specific amino acids, specific polypeptides, and polyene antibiotics.
The most logical substances for binding cholesterol would be those substances involved in cholesterol transport within biological systems. A number of papers discuss the isolation and character of these cholesterol carrier proteins. Examples of such papers include:
In "The Role of a Carrier Protein in Cholesterol and Steroid Hormone Synthesis by Adrenal Enzymes 1, 2, "Kan et al., Biochemical and Biophysical Research Communications, 48(2), 423-429 (1972). The adrenal glands are shown to contain a sterol carrier protein (SCP) similar to that of liver-SCP. The paper points out that SCP is required for cholesterol synthesis from squalene and steroid synthesis from cholesterol. SCP is thought to be present in yeast and protozoa.
Takase et al., "Characterization of Sterol Carrier Protein Binding with 7-Dehydrocholesterol and Vitamin D", J. Nutr. Sci. Vitaminol., 23, 53-61, (1977) discusses the role of Vitamin D3 in the relationship between rat liver sterol carrier protein (SCP) and cholesterol.
LeFevre et al., "Adrenal Cholesterol-Binding Protein: Properties and Partial Purification", Febs. Letters, 89(2), 287-292 (1978) discusses a heat-stable protein (CPB) present in the cytosol of adrenal glands, testes and ovaries which specifically binds tritiated cholesterol. A case is made differentiating the CPB from the known sterol-carrier protein present in liver.
Higuchi et al., "Comparative Studies on a Heat-Stable Cholesterol-Binding Protein in Dental Cyst Fluid and Serum", Int. J. Biochem., 13, 777-782 (1981) presents data indicating that dental cyst fluid contains a heat-stable cholesterol-binding protein (CPB) factor. A heated supernatant fraction of cyst fluid is reacted with a C.sup.14 labeled cholesterol. A SEPHADEX column is used to separate the bound cholesterol from the free cholesterol. The concentration of bound cholesterol is determined by plotting the radioactivity.
Chen et al., "Prostate Protein: Isolation and Characterization of the Polypeptide Components and Cholesterol Binding", J. Biol. Chem., 257(1), 116-121 (1982) presents data concerning .alpha.-protein, a major protein in rat prostate secretions which originates in the rat ventral prostate o-Protein is shown to bind cholesterol.
Sziegoleit, "Purification and Characterization of a Cholesterol-Binding Protein from Human Pancreas," Biochem. J., 207, 573-582 (1982) describes a cholesterol binding protein discovered in excreted lavage fluids. Immunologic analysis of the gut specific protein shows the organ of origination to be the pancreas. The protein not only binds cholesterol, but also the bile salt deoxycholate. The protein comprises a single polypeptide chain having a molecular weight of 28,000. The isoelectric point is at pH 4.9.
Regenass-Klotz et al., "Specific Binding of cholesterol to Chromatin Prepared from Mouse Spleen Cells", Can. J. Biochem. Cell Biol., 62, 94-99 (1984) presents data showing that cholesterol specifically binds to the chromatin of mouse splenic lymphocytes. The evidence points to the cholesterol actually binding to a high molecular weight protein in the chromatin preparation and not to deoxyribonucleic acid.
The carrier proteins discussed above show great affinity for cholesterol and would theoretically provide specific ligands for affinity chromatography; the binding site of any of these proteins could be immobilized and used for liquid chromatography to specifically remove cholesterol. However, the binding site is only a small part of the protein molecule, and thus a large mass of protein would be required to remove a small amount of cholesterol. In addition, if the natural protein is employed, the possibility of contaminants causing hepatitis and other viral diseases is always present. Consequently, in practice these methods are entirely unacceptable for use in food processing.
Klimov et al., "Interaction of Cholesterol with Polypeptides and Amino Acids", documents certain binding sites on amino acids and polypeptides which bind cholesterol. This article teaches that amino acids and compounds containing guanidinio groups (e.g., guanidine, metformine, arginine and polyarginine) and gamma-amino groups (e.g., lysine and polylysine) bind to cholesterol; however, there is no suggestion for using these substances for the extraction of cholesterol.
Certain antibiotics have been noted for their ability to bind to cholesterol. Notable amongst these are the polyenes filipin and pimaricin. Bornig et al., "Staining of Cholesterol with the Fluorescent Antibiotic Filipin", Acta Histochem., 50, 110-115 (1974) documents the affinity of filipin for non-esterified cholesterol, and cited its utility as a histochemical stain. Patterson, "Effects of Experimental Conditions on the Interaction of Filipin and Pimaricin with Cholesterol", Antibiot. (Tokyo), 32(11), 1193-2000 (1979) documents pimaricin as having similar properties to filipin. Others have noted the affinity that these polyenes have for cholesterol; see, for example,
Geyer et al., "Filipin--A Histochemical Fluorochrome for Cholesterol", Acta Histochem [Suppl] (Jena), 15, 207-212 (1975);
Bittman et al., "Determination of Cholesterol Asymmetry by Rapid Kinetics of Filipin-Cholesterol Association: Effect of Modification in Lipids and Proteins", Biochemistry, 20(9), 2425-2432 (1981); and
Behnke et al., "Filipin as a Cholesterol Probe. II. Filipin Cholesterol Interaction in Red Blood Cell Membranes", Eur. J. Cell Biol., 35(2), 200-215 (1984). None of the references suggest the use of pimaricin or filipin as a ligand to remove cholesterol from foodstuffs.
U.S. Pat. No. 4,297,220 to Meitzner et al. (assigned to Rohm and Haas) describes a method for adsorbing an organic material from a fluid or fluid mixture containing the same which comprises contacting the fluid or fluid mixture containing organic material with a macroreticulated crosslinked copolymer having a plurality of microscopic channels resulting from liquid expulsion of a precipitating agent during polymerization of a monomer mixture under suspension polymerization conditions in an aqueous media of (1) a polyvinylidene monomer containing a plurality of ethylenically unsaturated groups in a non-conjugated relationship and (2) at least one monoethylenically unsaturated monomer, said copolymerization taking place in the pressure [sic] of a liquid which is a solvent for the monomer mixture and which does not swell the copolymer resulting from the copolymerization, the liquid being present in an amount sufficient to cause separation of the copolymer from the monomer phase, whereby the organic material is adsorbed by the macroreticulated crosslinked copolymer. The solvent in the polymerization mixture causes the formation of microscopic channels in the copolymer; the purpose of these channels appears to be to facilitate access of the organic material to the ion-exchange centers which are later formed on the copolymer.
U.S. Pat. No. 4,454,056 to Kittelmann et al. describes a process for the modification of the surfaces of zeolites with organosilanes which have at least one alkoxy group bonded to the silicon atom of the silane. The purpose of the surface modification is to prevent agglomeration of the zeolite when it is mixed with a detergent, so that the resultant mixture will remain free-flowing in use.
U.S. Pat. No. 4,637,861 to Krull et al. describes a stabilized, lipid-membrane based device comprising a perturbable lipid membrane and a membrane-stabilizing support. The lipid membrane includes a complexing agent for selectively interacting with a specified chemical species to perturb the lipid membrane, and each membrane-forming lipid includes a polar head group, a first long chain through which the lipid is anchored to a binding site on the support, and a fluidity-providing second chain, which is not attached to the support. The binding sites on the support of spaced apart so as to provide a lipid packing density that permits axial rotation of each membrane-forming lipid about its long, anchored chain, and is yet sufficiently close to provide a high ion impermeability to the unperturbed lipid membrane, but an increased permeability when disturbed. The lipid is preferably anchored to the support via a silicon atom (see column 4, lines 35-42 of the patent).
U.S Pat. No. 4,681,870 to Balint et al. describes a method for preparing an immunoadsorbent material useful for removing IgG and IgG-complexes from biological fluids, this method comprising introducing free amino or carboxyl groups on to a silica matrix, reacting the silica matrix with purified protein A in the presence of a carbodiimide at a pH of 3.5 to 4.5 to covalently link the protein A to the silica matrix through the amino or carboxyl groups, and washing the silica matrix at pH 2.0 to 2.5 to remove loosely bound protein A. In effect, this method simply bonds the protein A, which effects the actual conjugation with the IgG, to a support, thereby providing a solid, insoluble form of the protein A able to function as the active material of a column through which liquid to be stripped of IgG can be run.
U.S. Pat. No. 4,665,110 to Zones describes a process for the synthesis of zeolites using adamantane compounds as templating agents.
Lok et al., "The role of organic molecules in molecular sieve synthesis", Zeolites, 3, 282 (1983), is a review article discussing the relationship between the templating agents used in the synthesis of zeolites, aluminophosphates and similar compounds, and the structures of the molecular sieves produced.
Lehn, "Supramolecular chemistry: receptors, catalysts and carriers", Science, 227, 849 (1985) is a general review of ways in which polymolecular complexes are formed, and includes a discussion of various types of binding by polydentate ligands.
Wehr, "Sample preparation and column regeneration in biopolymer separations", J. Chrom. 418, 27 (1987) discusses the use of selective sorbents, including silica-based sorbents, for selective adsorption and fractionation of polypeptides from complex biological mixtures.
Evershed et al., "Strategy for the analysis of steryl esters from plant and animal tissues", J. Chrom. 400, 187 (1987), discusses various techniques for separation of steryl esters from complex biological mixtures, including various chromatographic separations.
Chemical Abstracts, 108, 112,170k and 150,453n, describe polydentate ligands and cavitands capable of binding metal ions and small guest molecules. There is no disclosure of separation of cholesterol or any closely related material.
Yamamura et al., Guest Selective Molecular Recognition by an Octadecylsilyl Monolayer Covalently Bound on an SnO.sub.2 Electrode, J. Chem. Soc., Chem. Commun., 1988, 79-81 discloses the technique of adsorbing a templating molecule, such as a cholesterol derivative, on to a tin oxide surface, modifying the surface using a silane derivative and desorbing the templating molecule in order to provide a modified surface having cavities which will accommodate cholesterol or other molecules which it is desired to adsorb. This paper demonstrates that, when the modified surface is placed in contact with a solution containing cholesterol, access of Vitamin K.sub.1 to the electrode is blocked; however, the paper does not indicate how much cholesterol is adsorbed by the modified surface, nor does it suggest that such a modified surface can be used to separate cholesterol from fluid mixtures.
Chemical Abstracts, 108, 127,097r, is an abstract of a more recent paper by the same researchers as the previous paper, and records the use of ODS-modified silica for the adsorption of artificial liposomes.
It has now been discovered that sterol compounds can be removed from fluid mixtures by contacting the mixture with a surface-modified adsorbent. In particular, sterol compounds can be removed from liquid comestible mixtures, including foodstuffs, in a highly selective manner and without substantial changes in the physical and nutritional properties of the foodstuff or other liquid comestible mixture by contacting the mixture with a surface-modified adsorbent. Also, as already indicated, the same surface-modified adsorbents can be used to remove sterol compounds from solvents, such as liquid or supercritical carbon dioxide, which have been used for extraction of foodstuffs, this removal of sterol compounds being effected without the need to evaporate and reliquify the solvent.